The major defect in type 2 diabetes is insulin resistance of peripheral tissues, primarily muscle and adipose tissue. Insulin-stimulated glucose uptake is mediated by the insulin-responsive glucose transporter, (GLUT4), GLUT4 vesicle translocation and fusion with the plasma membrane are critical for glucose homeostasis. In order for vesicle fusion to occur, the cortical cytoskeletal networks at the plasma membrane have to be disrupted in a temporal and spatial manner. While recent studies have established that actin reorganization is required for GLUT4 vesicle translocation and membrane remodeling, little is known about the motor forces required for these processes. The conventional nonmuscle myosin, myosin II is an actin-based motor protein that has been shown to facilitate vesicle trafficking in various cell types. The goal of this proposal is to characterize the role of myosin II in insulin-mediated GLUT4 vesicle trafficking in adipocytes by identifying the myosin II isoform responsible for regulating GLUT4 trafficking and determining its mode of action and regulation. We will use the 3T3-L1 adipocyte cell culture model to investigate the role of myosin II in GLUT4 trafficking since much of what is known about insulin-stimulated glucose uptake in adipocytes has been elucidated using this cell line. Our preliminary studies show that inhibition of myosin II impairs GLUT4- mediated glucose uptake but not GLUT4 translocation to the plasma membrane. We also show that adipocytes express both myosin IIA and IIB isoforms and that myosin IIA is recruited to the plasma membrane upon insulin stimulation. Based on our findings, we hypothesize that myosin II is activated upon insulin stimulation and translocates to the cell cortex to facilitate GLUT4 fusion with the plasma membrane. This project lends itself extremely well to student involvement. Undergraduate students are exposed to the fundamental concepts of hormone action, vesicle transport and glucose metabolism early in the Biology curriculum. This project will allow students to gain practical laboratory experience of these concepts using standard molecular, cellular and biochemical techniques. This project also allows students to integrate key concepts taught in the classroom to address a physiologically relevant question in the laboratory. Thus the overall aim of this proposal is to gain insight on the factors regulating the dynamic reorganization of the cytoskeleton during insulin-stimulated GLUT4 vesicle trafficking in order to provide potential pharmaceutical targets for drug therapies aimed at restoring insulin sensitivity and glucose homeostasis. [unreadable] [unreadable]